The present invention relates to nucleic acid sequences and methods useful for producing recombinant glucose-6-phosphatase (G-6-Pase). In addition, the present invention relates to specific mutations in the gene encoding human G-6-Pase and methods for detecting the mutations and thus diagnosing the genetic disease that causes glycogen storage disease type 1A.
Glucose-6-phosphatase catalyzes the terminal reaction in gluconeogenesis and glycogenolysis and is integral to the endoplasmic and nuclear membrane. The enzyme is thought to be arranged in the native microsomal membrane in such a way that it is not directly accessible to its substrate as well as inhibitors of low molecular mass nor to antibodies or proteases from the cytoplasmic surface. The enzyme may traverse the microsomal membrane as an integral channel protein. The catalytic region of G-6-Pase is theorized as being part of a channel which is maintained by the native conformation of the enzyme that in turn is elicited by the intimate microenvironment of the membrane. With this in mind, the specificity for glucose-6-phosphatase, development of latency, the high thermal sensitivity induced by detergent treatment of the native membrane, and the kinetic response to chemical modifying agents are considered to be properties of the enzyme itself, and not secondary reactions resulting from membrane disruption.
The solubilization of the enzyme from its natural lipid environment leads to rapid and irreversible inactivation and as a result has hindered purification. The purification of a detergent-soluble pyridoxylated G-6-Pase from rat liver was reported by Speth, M. and H.-U. Schulze in Eur. J. Biolchem., 208:643-650 (1992), incorporated herein by reference. The 700-fold purification was achieved by covalently labeling of the enzyme in native rat liver microsomes with pyridoxal 5'-phosphate and NaBH.sub.4, followed by solubilization of the microsomes with Triton X-100, chromatography on phenyl-Sepharose, hydroxyapatite, DEAE-Sephacel and a second chromatography step on hydroxyapatite. Analysis of the purified enzyme on SDS/PAGE showed a band migrating at 35-kDa. Purification was achieved due to the interaction between the negatively charged enzyme-bound phosphate label of the pyridoxylated G-6-Pase and the Ca.sup.+ of the hydroxyapatite resin.
Glycogen metabolism in the liver plays a major role in the homeostatic regulation of blood glucose levels. The synthesis and degradation of glycogen are tightly regulated by homeostatic and hormonal mechanisms which ensure an optimal utilization of the polysaccharide.
Glycogen storage diseases are known to be the result of at least 10 different genetic defects within the group of enzymes and transport proteins required by glycogen metabolism. Glycogen storage disease Type Ia (GSD, also known as yon Gierke disease) is defined as the deficiency of glucose-6-phosphatase which is normally present in liver, kidney, and intestine. In a subgroup of the disease, types 1b, 1c and 1d, the putative cause is the deficiency of three transport proteins termed T1, T2 and T3, respectively. These transport proteins allow the substrates and products, glucose-6-phosphate, phosphate (and pyrophosphate) and glucose to cross the endoplasmic reticulum membrane. Glycogen storage disease type la is inherited by 1 in 100,000 to 300,000 as an autosomal recessive trait and is usually manifested during the first 12 months of life by symptomatic hypoglycemia, or by the recognition of hepatomegaly. In addition, GSD type 1a can have indications of growth retardation, delayed adolescence, lacticacidemia, hyperlipidemia, hyperuricemia, and in adults, hepatic adenomas.
While it is known that GSD type 1a is due to inactive G-6-Pase and the cause of this inactivity most likely genetic, the specific genetic alterations are unknown. Knowledge of the gene sequence encoding G-6-Pase as well as the mutated gene sequences that result in inactive G-6-Pase is an important and basic discovery toward understanding the molecular basis of this disorder and for developing diagnostic tools and therapeutic treatments. To these ends the present invention disclosing the gene sequence of human as well as murine G-6-Pase (Seq. ID No.:35) and mutations that result in inactive human G-6-Pase is presented.